Journal: Proteomics

Article Title: Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin‐3

doi: 10.1002/pmic.201500501

Figure Lengend Snippet: Identification of proteins with the most differential change at 40 and 80% cell density in response to Nutlin‐3 treatment. (A) Identification of proteins perturbed by Nutlin‐3 using SWATH MS. HCT116 cells were incubated for 4 h with Nutlin‐3 under conditions in which MDM2 is just beginning to be stabilized (Fig. B). This ensures that we capture changes in the cellular proteome just when MDM2 is starting to be perturbed in cells by Nutlin‐3. The cell pellets were processed using MS SWATH to identify differentially perturbed proteins (Supporting Information Tables 1 and 2), some of which are highlighted (in green) as a function of 40 or 80% cell density. (B) A scatter plot of the total protein changes as a function of cell density and fold change (log 2 ) with mitochondrial proteins highlighted in red.

Article Snippet: The cells were fixed onto slides with 4% paraformaldehyde in PBS for 20 min at RT, permeabilized for 10 min in 0.25% Triton x‐100 in PBS and blocked with 3% BSA in PBS for 30 min. Antibodies from different species were then incubated on the slides, with combinations of MDM2 mouse mAb (4B2) with either rabbit pAb DLD (sc‐135027; Santa Cruz) or p53 (CM‐1), at a 1:250 dilution for 1 h at RT.

Techniques: Data-independent acquisition, Incubation

Journal: Proteomics

Article Title: Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin‐3

doi: 10.1002/pmic.201500501

Figure Lengend Snippet: The allosteric effect of Nutlin‐3 on MDM2 functions. (A) MDM2 has multiple functional domains including an N‐terminal peptide‐binding groove that is the binding site of Nutlin‐3 ; a central domain that responds allosterically to N‐terminal MDM2‐binding ligands (like Nutlin‐3) to increase p53 binding and ubiquitination (arrows) , and a RING domain that is required for E2 docking and allosteric control of ubiquitination by the E2, UBCH5 . N‐terminal domain Nutlin‐3 binding by MDM2 can stimulate p53 ubiquitination , stimulate ubiquitin‐dependent protein activation of Notch , and direct binding to alter target protein oligomerization of NPM . Thus, Nutlin‐3 can dissociate or induce various protein–protein interactions due to the allosteric effects of ligands on MDM2 function. These data suggest a complex effect of MDM2 ligands on changes in the steady‐state cellular proteome. (B–D) HCT116 cells (p53+ and p53‐null, as indicated) where grown to 40 and 80% density as defined in the methods. Cells were treated with Nutlin‐3 (20 μM final concentration) or DMSO control and after 4 h cells were harvested for lysis without proteasome inhibitor treatment (that would artificially elevate levels of target protein). Lysates were blotted with the indicated antibodies (MDM2, p53, and tubulin as a loading control). The data demonstrate that p53 protein is induced equivalently by Nutlin‐3 at either cell density, but MDM2 protein exhibits higher steady‐state levels at the lower density after Nutlin‐3 treatment, as reported previously .

Article Snippet: The cells were fixed onto slides with 4% paraformaldehyde in PBS for 20 min at RT, permeabilized for 10 min in 0.25% Triton x‐100 in PBS and blocked with 3% BSA in PBS for 30 min. Antibodies from different species were then incubated on the slides, with combinations of MDM2 mouse mAb (4B2) with either rabbit pAb DLD (sc‐135027; Santa Cruz) or p53 (CM‐1), at a 1:250 dilution for 1 h at RT.

Techniques: Functional Assay, Binding Assay, Ubiquitin Proteomics, Control, Activation Assay, Protein-Protein interactions, Concentration Assay, Lysis

Journal: Proteomics

Article Title: Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin‐3

doi: 10.1002/pmic.201500501

Figure Lengend Snippet: Immunochemical analysis of dihydrolipoamide dehydrogenase protein levels after MDM2 perturbation. (A and B) HCT116 cells (p53+) where grown to 40 or 80% density. Cells were treated with Nutlin‐3 (20 μM final concentration) or DMSO control and after 4 h, cells were harvested for lysis without proteasome inhibitor treatment that would artificially elevate levels of target protein. Lysates were blotted with the indicated antibodies to dihydrolipoamide dehydrogenase and tubulin as a loading control. (C–F). HCT116 cells grown at 80% density and then were treated with control siRNA (C) or siRNA to deplete MDM2 (M) for 24 h, followed by treatment with DMSO or Nutlin‐3. The lysates were then immunoblotted for p53, MDM2, and dihydrolipoamide dehydrogenase, and the loading control.

Article Snippet: The cells were fixed onto slides with 4% paraformaldehyde in PBS for 20 min at RT, permeabilized for 10 min in 0.25% Triton x‐100 in PBS and blocked with 3% BSA in PBS for 30 min. Antibodies from different species were then incubated on the slides, with combinations of MDM2 mouse mAb (4B2) with either rabbit pAb DLD (sc‐135027; Santa Cruz) or p53 (CM‐1), at a 1:250 dilution for 1 h at RT.

Techniques: Concentration Assay, Control, Lysis

Journal: Proteomics

Article Title: Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin‐3

doi: 10.1002/pmic.201500501

Figure Lengend Snippet: Nutlin‐3 dissociates dihydrolipoamide dehydrogenase and dihydrolipoamide acetyltransferase protein–protein interactions within the pyruvate dehydrogenase holoenzyme complex. (A–F) HCT116 cells were treated with DMSO or Nutlin‐3 (20 μM) for 4 h. Cells were fixed and processed for proximity ligation as recorded in the Methods using antibodies to dihydrolipoamide dehydrogenase (mouse) and dihydrolipoamide acetyltransferase (rabbit). Cells were imaged using FITC as a readout of proximity ligation (representative images in A, B, and C), DAPI plus merged FITC (D, E, and F). (A, D) DMSO control; (B, E) Nutlin‐3 effects; (C, F) DMSO with BSA controls without primary antibodies (G and H). MDM2:p53 complexes were evaluated with DMSO control (G) or with Nutlin‐3 (H; representative images are a merge of FITC proximity ligation and DAPI to localize the nucleus). The quantitation of the average dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase foci in cells is summarized in I (using ImageJ software).

Article Snippet: The cells were fixed onto slides with 4% paraformaldehyde in PBS for 20 min at RT, permeabilized for 10 min in 0.25% Triton x‐100 in PBS and blocked with 3% BSA in PBS for 30 min. Antibodies from different species were then incubated on the slides, with combinations of MDM2 mouse mAb (4B2) with either rabbit pAb DLD (sc‐135027; Santa Cruz) or p53 (CM‐1), at a 1:250 dilution for 1 h at RT.

Techniques: Protein-Protein interactions, Ligation, Control, Quantitation Assay, Software

Journal: Proteomics

Article Title: Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin‐3

doi: 10.1002/pmic.201500501

Figure Lengend Snippet: The effects of Nutlin‐3 on dihydrolipoamide dehydrogenase and MDM2 complexes protein in vitro and in vivo. (A and B) The effects of Nutlin‐3 on MDM2 protein–protein interactions. An ELISA was used to measure the binding of MDM2 to A, dihydrolipoamide dehydrogenase or B, p53 . The purity of the indicated proteins measure by Coomassie blue is in Supporting Information Fig. 2. Target protein p53 or dihydrolipoamide dehydrogenase was coated onto the solid phase as indicated in the Methods. Ligand‐free MDM2 (DMSO control) or Nutlin‐3 (20 μM in DMSO) bound MDM2 was titrated into reactions followed by immunochemical quantitation of the amounts of MDM2 bound. MDM2 activity is depicted in relative light units as a function of increasing MDM2 protein levels (in nanograms). (C–H) In vivo binding of MDM2 and dihydrolipoamide dehydrogenase. Proximity ligation assays were used according to the Methods to measure MDM2 and dihydrolipoamide dehydrogenase binding in situ. (C and D) FITC and DAPI merged images, respectively, in proliferating cells treated with DMSO control. (E and F) FITC and DAPI merged images, respectively, in cells treated with Nutlin‐3 (20 μM) for 4 h. (G and H) FITC and DAPI merged images, respectively, in proliferating cells treated with DMSO control using BSA in place of primary antibodies as a negative control. (I and J) Total cellular distribution of MDM2 and dihydrolipoamide dehydrogenase. Immunofluorescence was used with specific antibodies in fixed cells according to the Methods to measure the total distribution of MDM2 and dihydrolipoamide dehydrogenase pools in the absence or presence of Nutlin‐3 (20 μM). (K) Quantitation of MDM2 and dihydrolipoamide dehydrogenase protein–protein interaction foci in the absence and presence of Nutlin‐3 using proximity ligation .

Article Snippet: The cells were fixed onto slides with 4% paraformaldehyde in PBS for 20 min at RT, permeabilized for 10 min in 0.25% Triton x‐100 in PBS and blocked with 3% BSA in PBS for 30 min. Antibodies from different species were then incubated on the slides, with combinations of MDM2 mouse mAb (4B2) with either rabbit pAb DLD (sc‐135027; Santa Cruz) or p53 (CM‐1), at a 1:250 dilution for 1 h at RT.

Techniques: In Vitro, In Vivo, Protein-Protein interactions, Enzyme-linked Immunosorbent Assay, Binding Assay, Control, Quantitation Assay, Activity Assay, Ligation, In Situ, Negative Control, Immunofluorescence

Journal: Proteomics

Article Title: Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin‐3

doi: 10.1002/pmic.201500501

Figure Lengend Snippet: The effects of Leptomycin B on dihydrolipoamide dehydrogenase and MDM2 complexes in cells using proximity ligation assays. (A and B) The effects of Leptomycin B after four hours of treatment on MDM2 and dihydrolipoamide dehydrogenase protein–protein interactions with images depicting FITC proximity ligation [24] (A) and DAPI nuclear stain (in blue) and MitoTracker Red CMXros (in red) as a merged image (B). (C and D) The effects of Nutlin‐3 on MDM2 and dihydrolipoamide dehydrogenase protein–protein interactions with images depicting FITC proximity ligation (C) and DAPI nuclear stain (in blue) and MitoTracker Red CMXros (in red) as a merged image (D). (E and F) The effects of Leptomycin B and Nutlin‐3 combined on MDM2 and dihydrolipoamide dehydrogenase protein–protein interactions with images depicting FITC proximity ligation (E) and DAPI nuclear stain (in blue) and MitoTracker Red CMXros (in red) as a merged image (F). (G) Quantitation of protein–protein interaction foci with the indicated treatment with DMSO, Leptomycin B, Nutlin‐3, or Nutlin‐3, and Leptomycin B combined.

Article Snippet: The cells were fixed onto slides with 4% paraformaldehyde in PBS for 20 min at RT, permeabilized for 10 min in 0.25% Triton x‐100 in PBS and blocked with 3% BSA in PBS for 30 min. Antibodies from different species were then incubated on the slides, with combinations of MDM2 mouse mAb (4B2) with either rabbit pAb DLD (sc‐135027; Santa Cruz) or p53 (CM‐1), at a 1:250 dilution for 1 h at RT.

Techniques: Ligation, Protein-Protein interactions, Staining, Quantitation Assay

Journal: Proteomics

Article Title: Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin‐3

doi: 10.1002/pmic.201500501

Figure Lengend Snippet: Model summarizing the effects of Nutlin‐3 on dihydrolipoamide dehydrogenase and MDM2 localizations. (Left panel) In proliferating cells, there are at least two pools of dihydrolipoamide dehydrogenase. One pool interacts predominantly with components of the pyruvate dehydrogenase complex and the second pool with MDM2, predominantly in the cytosol. (Right panel) Following Nutlin‐3 treatment, perturbation of the mitochondrial proteome results in dissociation of the dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase, suggesting a disruption of pyruvate dehydrogenase holoenzyme complex. In addition, although minimal dihydrolipoamide dehydrogenase is observed in the nucleus in untreated cells, the Nutlin‐3‐dependent import of MDM2 into the nucleus coincides with increased dihydrolipoamide dehydrogenase: MDM2 complexes in the nucleus. It remains to be determined if the binding of dihydrolipoamide dehydrogenase to MDM2 in the nucleus has a direct effect on p53 protein activation.

Article Snippet: The cells were fixed onto slides with 4% paraformaldehyde in PBS for 20 min at RT, permeabilized for 10 min in 0.25% Triton x‐100 in PBS and blocked with 3% BSA in PBS for 30 min. Antibodies from different species were then incubated on the slides, with combinations of MDM2 mouse mAb (4B2) with either rabbit pAb DLD (sc‐135027; Santa Cruz) or p53 (CM‐1), at a 1:250 dilution for 1 h at RT.

Techniques: Disruption, Binding Assay, Activation Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of STAT5 induces G1 cell cycle arrest and reduces tumor cell invasion in human colorectal cancer cells.

doi: 10.1038/labinvest.2009.11

Figure Lengend Snippet: Figure 1 RNAi treatment decreases STAT5 signaling in CRC cells. (a) Western blot analysis demonstrated that both STAT5 and pSTAT5 were depleted in SW1116 cells after transfection with Dharmacon STAT5 siRNA. The lowest levels of STAT5 expression were detected on day 3, and the proteins were recovered at day 4 post-transfection. (b) At 72 h post- transfection, western blot analysis showed that STAT5 siRNA (Santa Cruz) induced a downregulation of STAT5 and pSTAT5 in CRC cells. In addition, STAT5 siRNA-induced alterations in several, but not all downstream targets of STATs in CRC cells at 72 h after treatment. Bcl-2 protein was downregulated simultaneous to an upregulation of p16ink4a, p21waf1/cip1

Article Snippet: All antibodies in this study were purchased from Cell Signaling Technology Inc. (USA), except the following antibodies: phospho-STAT5 (pSTAT5Tyr694/Tyr699) (Santa Cruz, CA, USA), Bcl-2 (R&D, USA) and GAPDH (Kangchen, China).

Techniques: Western Blot, Transfection, Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of STAT5 induces G1 cell cycle arrest and reduces tumor cell invasion in human colorectal cancer cells.

doi: 10.1038/labinvest.2009.11

Figure Lengend Snippet: Figure 2 The functional role of STAT5 signaling on CRC cell growth and cell cycle progression. (a) Cell viability was determined by the CCK-8 assay following treatment of CRC cells with STAT5 siRNAs. The percentage of viable cells was determined as described in the Materials and Methods section. STAT5 siRNA inhibited CRC cell growth. This suppression persisted for 72 h, after which the cell recovered at 96 h post-transfection. The results represent mean±s.d. of three experiments. (b) Cell cycle analysis was performed after treating CRC cells with STAT5 siRNA (Santa Cruz). STAT5 knockdown led to a block in the cell cycle at the G1 phase at 72 h after transfection.

Article Snippet: All antibodies in this study were purchased from Cell Signaling Technology Inc. (USA), except the following antibodies: phospho-STAT5 (pSTAT5Tyr694/Tyr699) (Santa Cruz, CA, USA), Bcl-2 (R&D, USA) and GAPDH (Kangchen, China).

Techniques: Functional Assay, CCK-8 Assay, Transfection, Cell Cycle Assay, Knockdown, Blocking Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of STAT5 induces G1 cell cycle arrest and reduces tumor cell invasion in human colorectal cancer cells.

doi: 10.1038/labinvest.2009.11

Figure Lengend Snippet: Figure 3 The functional role of STAT5 signaling on the invasive ability of CRC cells. (a) CRC cells transfected with STAT5 siRNA were examined for their invasive capability. At 48 h post-transfection, the number of migrated cells significantly decreased compared with untreated cells (*Po0.05). (b) Western blot analysis showed that STAT5 siRNA (Santa Cruz) induced an upregulation of E-cadherin, simultaneous with a downregulation of FAK in CRC cells at 72 h post-transfection of the cells. (c) Effects of STAT5 siRNA on the secretion of VEGF, MMP2 and MMP9. At 48 h post-transfection, the concentrations of VEGF and MMP2 were decreased compared with that of untreated cells (*Po0.05). The experiment was performed three times with consistent findings.

Article Snippet: All antibodies in this study were purchased from Cell Signaling Technology Inc. (USA), except the following antibodies: phospho-STAT5 (pSTAT5Tyr694/Tyr699) (Santa Cruz, CA, USA), Bcl-2 (R&D, USA) and GAPDH (Kangchen, China).

Techniques: Functional Assay, Transfection, Western Blot

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of STAT5 induces G1 cell cycle arrest and reduces tumor cell invasion in human colorectal cancer cells.

doi: 10.1038/labinvest.2009.11

Figure Lengend Snippet: Figure 4 Immunohistochemical staining of the tissue microarray. Predominantly cytoplasmic staining of STAT5 was detected in normal colonic epithelium, adenomas and primary colon adenocarcinomas (as indicated by red arrows). pSTAT5 localized to the cytoplasm of adenoma cells and colon adenocarcinoma cells, although predominantly found in the nucleus of normal epithelium cells (Black arrows indicate pSTAT5 protein is shuttled from nucleus to cytoplasm in the oncogenesis of CRC; magnification ¼ 400).

Article Snippet: All antibodies in this study were purchased from Cell Signaling Technology Inc. (USA), except the following antibodies: phospho-STAT5 (pSTAT5Tyr694/Tyr699) (Santa Cruz, CA, USA), Bcl-2 (R&D, USA) and GAPDH (Kangchen, China).

Techniques: Immunohistochemical staining, Staining, Microarray

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Inhibition of STAT5 induces G1 cell cycle arrest and reduces tumor cell invasion in human colorectal cancer cells.

doi: 10.1038/labinvest.2009.11

Figure Lengend Snippet: Figure 5 Interaction of STAT5 with MAPK, PI3K and AKT in CRC cells. (a) Protein–protein interactions were determined by co-immunoprecipitation analysis. Co-immunoprecipitation analysis revealed that STAT5 formed a complex with p44/42 MAPK and SAPK/JNK in SW1116 cells. However, no significant interactions of STAT5 with PI3K, p38, AKT were detected in our studies. (b) Reciprocal immunoprecipitations were performed with antibodies directed against p44/42 or SAPK/JNK or a non-relevant antibody (purified human IgG, negative control), then detected STAT5 by western blot. These results also validate the interactions of STAT5 with p44/42 MAPK or SAPK/JNK in CRC cells.

Article Snippet: All antibodies in this study were purchased from Cell Signaling Technology Inc. (USA), except the following antibodies: phospho-STAT5 (pSTAT5Tyr694/Tyr699) (Santa Cruz, CA, USA), Bcl-2 (R&D, USA) and GAPDH (Kangchen, China).

Techniques: Protein-Protein interactions, Immunoprecipitation, Purification, Negative Control, Western Blot

Journal: Biology of reproduction

Article Title: Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

doi: 10.1095/biolreprod.102.011387

Figure Lengend Snippet: FIG. 1. A schematic drawing illustrates the four signaling pathways utilized by TGFb to affect cellular function. This diagram was prepared based on reviews [18, 19, 21]. Original articles that describe the discovery of the key molecules depicted in these pathways can be found in these reviews. MEKKs, MAP/ERK kinase kinases, which include MEKK1, MEKK2, MEKK3, and others; JNK, c-Jun NH2-terminal kinase also known as Jun kinase or stress-activated protein kinase, SAPK; JNKK, c-Jun NH2-terminal kinase kinase; MKK3, MAP kinase kinase 3; MEK1/2, MAP/ERK kinase 1 and MAP/ERK kinase 2; ERK1/2, extracellular signal-regulated kinase 1 and extracellular signal-regulated kinase 2.

Article Snippet: Antibodies against TGFb3 (cat. no. sc-82, lot. no. H280), MEKK2 (cat. no. sc-1088, lot. no. A181), Rac2 (cat. no. sc-96, lot. no. G231), and N-Ras (cat. no. sc-31, lot. no. D111) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Protein-Protein interactions, Cell Function Assay

Journal: Biology of reproduction

Article Title: Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

doi: 10.1095/biolreprod.102.011387

Figure Lengend Snippet: FIG. 4. Relative expression of the TGFb- upstream signal transducers in Sertoli and germ cells isolated from 20-day-old rat testes. A) Semiquantitative RT-PCR was performed to assess the steady-state mRNA levels of Smad2, Cdc42, Rac2, MEKK2, and N-Ras in Sertoli and germ cells. Im- munoblots were performed in parallel ex- periments to assess the relative protein levels of these signal transducers in Sertoli and germ cells using the corresponding specific antibodies (see Materials and Methods). (C). B) This figure shows the corresponding densitometrically scanned results using autoradiograms or immuno- blots such as those shown in A and C. Re- sults are expressed as mean 6 SD using three batches of cells from three different experiments normalized against S16. Each experiment had triplicate cultures. These analyses revealed that results of immuno- blots are consistent with the RT-PCR data. Statistical analysis was performed by Stu- dent t-test comparing germ cells with the corresponding Sertoli cells, which was ar- bitrarily set at one. *, Significantly different from Sertoli cells by Student t-test, P , 0.05; **, significantly different from Sertoli cells by Student t-test, P , 0.01.

Article Snippet: Antibodies against TGFb3 (cat. no. sc-82, lot. no. H280), MEKK2 (cat. no. sc-1088, lot. no. A181), Rac2 (cat. no. sc-96, lot. no. G231), and N-Ras (cat. no. sc-31, lot. no. D111) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Biology of reproduction

Article Title: Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

doi: 10.1095/biolreprod.102.011387

Figure Lengend Snippet: FIG. 5. Developmental regulation of the steady-state mRNA levels of the TGFb up- stream signal transducers in Sertoli (A, B) and germ (C, D) cells. RT-PCR was per- formed to assess the steady-state mRNA levels of Smad2, Cdc42, Rac2, MEKK2, and N-Ras in Sertoli cells (A) and germ cells (C) during maturation. B, D) The cor- responding densitometrically scanned re- sults using autoradiograms such as those shown in A and C. Results are expressed as mean 6 SD using two batches of cells normalized against S16 from two different experiments. Each experiment had tripli- cate cultures. ns, Not significantly different from cultures isolated from rats at 20 days of age in B and 5 or 10 days of age in D, which was arbitrarily set at one, by Stu- dent t-test; *, significantly different by Stu- dent t-test, P , 0.05; **, significantly dif- ferent by Student t-test, P , 0.01; nd, not detectable.

Article Snippet: Antibodies against TGFb3 (cat. no. sc-82, lot. no. H280), MEKK2 (cat. no. sc-1088, lot. no. A181), Rac2 (cat. no. sc-96, lot. no. G231), and N-Ras (cat. no. sc-31, lot. no. D111) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation

Journal: Biology of reproduction

Article Title: Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

doi: 10.1095/biolreprod.102.011387

Figure Lengend Snippet: FIG. 6. Changes in the steady-state mRNA levels of TGFb upstream signal transducers in the testis during develop- ment. A) Semiquantitative RT-PCR was performed to assess the steady-state mRNA levels of Smad2, Cdc42, Rac2, MEKK2, and N-Ras in testes during maturation. B) This panel shows the corresponding densi- tometrically scanned results using autora- diograms such as those shown in A. Re- sults are expressed as mean 6 SD using testes from three different rats normalized against S16. ns, Not significantly different from rats at 5 days of age, which was arbi- trarily set at one, by Student t-test; *, signif- icantly different by Student t-test, P , 0.05; **, significantly different by Student t-test, P , 0.01.

Article Snippet: Antibodies against TGFb3 (cat. no. sc-82, lot. no. H280), MEKK2 (cat. no. sc-1088, lot. no. A181), Rac2 (cat. no. sc-96, lot. no. G231), and N-Ras (cat. no. sc-31, lot. no. D111) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Biology of reproduction

Article Title: Transforming growth factor beta3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway.

doi: 10.1095/biolreprod.102.011387

Figure Lengend Snippet: FIG. 8. Change in the steady-state mRNA and protein levels of the TGFb upstream signal transducers when the Sertoli cell TJ barrier was assembled in vitro in the ab- sence (control) and presence (test) of TGFb3. Sertoli cells (0.5 3 106 cells/cm2) cultured on Matrigel-coated dishes in the absence (A, C, E) or presence (B, D, F) of TGFb3 (3 ng/ml) were terminated by RNA STAT-60 or lysed in SDS sample buffer at specified time points. Cell lysates (;200 mg protein) from each time point were re- solved by SDS-PAGE under reducing con- ditions using 10% T SDS-polyacrylamide gels. Immunoblotting was performed to as- sess changes in the levels of Smad2, Cdc42, Rac2, N-Ras, and MEKK2 in the absence (A, E) and presence (B, F) of re- combinant TGFb3 (3 ng/ml). RT-PCR was performed to assess changes in the MEKK2 steady-state mRNA level in the absence (C) or presence (D) of recombinant TGFb3 (3 ng/ml). G, H) Corresponding densito- metrically scanned results using autoradio- grams and immunoblots shown in C–F. Results are expressed as mean 6 SD from three separate experiments using different batches of cells and normalized against S16. Each time point had duplicate cul- tures. ns, Not significantly different by AN- OVA, in which each sample at a given time point was compared with samples of all other time points within the same ex- perimental group; *, significantly different by ANOVA, P , 0.01; D, days.

Article Snippet: Antibodies against TGFb3 (cat. no. sc-82, lot. no. H280), MEKK2 (cat. no. sc-1088, lot. no. A181), Rac2 (cat. no. sc-96, lot. no. G231), and N-Ras (cat. no. sc-31, lot. no. D111) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: In Vitro, Control, Cell Culture, SDS Page, Western Blot, Reverse Transcription Polymerase Chain Reaction, Recombinant

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A Protein levels of YBX1 in 10 different types of tumors from the CPTAC database. B mRNA levels of YBX1 in 21 different types of tumors from the TCGA database. C–G Enrichment functional analysis of proteins interacting with YBX1 in ccRCC cell line 786-O through Co-IP pull-down and identified by mass spectrometry. C Mass spectrometry identification schematic. D Gene Ontology Biological Process (GO-BP) enrichment. E KEGG pathway enrichment. F UniPort annotation keywords. G Wiki Pathways enrichment analysis. ** P < 0.01, *** P < 0.001, **** P < 0.0001. Unpaired two-sided Student’s t -test in A and B. Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Functional Assay, Co-Immunoprecipitation Assay, Mass Spectrometry

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A Western blot validation of YBX1 protein expression upon knockdown and overexpression efficiency in ACHN and 786-O cells. B qRT-PCR validation of YBX1 mRNA expression upon knockdown and overexpression efficiency in ACHN and 786-O cells. C Lactate production upon YBX1 knockdown in ACHN and 786-O cells. D ATP production detection upon YBX1 knockdown in ACHN and 786-O cells. E Real-time monitoring of extracellular acidification rate (ECAR) in ACHN and 786-O cells after YBX1 knockdown, measured using a Seahorse Bioscience Analyzer. F Quantitative analysis of glycolytic capacity and glycolytic reserve. G Real-time monitoring of oxygen consumption rate (OCR) in ACHN and 786-O cells after YBX1 knockdown, measured using a Seahorse Bioscience Analyzer. H Quantitative analysis of mitochondrial respiratory capacity and respiratory reserve. I Lactate dehydrogenase (LDH) activity detection upon YBX1 knockdown and overexpression in ACHN and 786-O cells. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Unpaired two-sided Student’s t -test in B – D , F , H and I . Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Western Blot, Biomarker Discovery, Expressing, Knockdown, Over Expression, Quantitative RT-PCR, Activity Assay

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A , B Effects of YBX1 overexpression on the proliferation of ACHN and 786-O cells. A Colony formation assay. B CCK-8 cell proliferation assay. C, D Effects of YBX1 overexpression on the migration and invasion of ACHN and 786-O cells. C Transwell cell migration assay. D Transwell cell invasion assay. E Detection of bioluminescence intensity of 8 pairs of tumors in nude mice using an in vivo imaging system. F Quantification analysis of bioluminescence intensity at the site of renal orthotopic tumors. G Statistical analysis of in situ tumor weight, expressed as the weight of the left minus the corresponding right kidney of nude mice. H Immunohistochemical staining and quantification to detect protein expression of YBX1, LDHA and Ki-67 in tumor tissues from control and YBX1 knockdown groups in nude mice. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Unpaired two-sided Student’s t -test in A , C , D , F , G and H . Two-way ANOVA with correction for multiple comparisons in B. Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Over Expression, Colony Assay, CCK-8 Assay, Proliferation Assay, Migration, Cell Migration Assay, Invasion Assay, In Vivo Imaging, In Situ, Immunohistochemical staining, Staining, Expressing, Control, Knockdown

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A, B Protein expression levels of YBX1 ( A ) and LDHA ( B ) in ccRCC from the CPTAC database. C, D Correlation of gene expression between YBX1 and LDHA in ccRCC from the TCGA database ( C ) and in an Asian population ( D ). E Western blot analysis of YBX1 and LDHA protein expression in tumor (T) and paired adjacent non-tumor (P) tissues from 27 ccRCC patients, showing representative images from 4 pairs of tissues. F Quantification of YBX1 and LDHA proteins in ccRCC and adjacent non-cancerous tissues from 27 cases using Image J software, normalized to β-actin. G Correlation of YBX1 and LDHA protein expression in tumor tissues from 27 ccRCC patients. H-M Immunohistochemical staining to assess the expression of YBX1 and LDHA in 63 pairs of ccRCC and adjacent tissues. H, I Representative immunohistochemical staining images and quantification of IHC staining using Image J software. J Correlation of quantified immunohistochemical staining of YBX1 and LDHA in tumor tissues of 63 ccRCC patients. K-M Levels of YBX1 and LDHA in ccRCC patients stratified by tumor size, T stage and Fuhrman grade. N , O Impact of YBX1 expression on overall survival (OS) ( N ) and recurrence free survival (RFS) ( O ) in ccRCC patients from the TCGA database. ** P < 0.01, *** P < 0.001, **** P < 0.0001. Unpaired two-sided Student’s t -test in A , B , K , L and M . Spearman correlation statistics in C , D , G and J . Paired t -test in F and I. Log-rank test in N and O. Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Expressing, Gene Expression, Western Blot, Software, Immunohistochemical staining, Staining, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A Identification of LDHA as a potential YBX1-interacting protein by IP/MS analysis. B Protein-protein interaction (PPI) network diagram of YBX1 and LDHA from the GeneMANIA and STRING databases. C Co-IP assay to verify the YBX1-LDHA interaction in ccRCC cell lines. D Immunofluorescence staining to examine the expression of YBX1 and LDHA in ACHN and 786-O cell lines, with confocal microscopy used to observe the co-localization of YBX1 and LDHA. E Molecular docking analysis of the YBX1-LDHA interaction. F Schematic diagram of GFP-tagged YBX1 structural domain peptide fragments. G 786-O cells transfected with GFP-tagged YBX1 or its truncation mutants for 48 h, followed by Co-IP using anti-GFP antibody. H Schematic representation of Flag-tagged LDHA domain protein peptides. I 786-O cells transfected with Flag-tagged LDHA or its truncation mutants for 48 h, followed by Co-IP using anti-Flag antibody. J Assessment of the impact on LDH activity after transfecting YBX1 or its truncated mutants into 786-O cells for 48 h. ** P < 0.01, **** P < 0.0001, ns: no significant difference. One-way ANOVA with correction for multiple comparisons in J. Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Protein-Protein interactions, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Expressing, Confocal Microscopy, Transfection, Activity Assay

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A qRT-PCR analysis of LDHA mRNA expression levels in ACHN and 786-O cells following YBX1 knockdown and overexpression. B CUT&Tag peak plots and heatmap showing YBX1 binding intensity in ACHN cells. C Pie chart showing the distribution of YBX1 binding regions. D IGV plot showing the occupancy of YBX1 in the LDHA promoter region. E Motif diagram of YBX1 binding sequences from the JASPAR database. F Molecular docking analysis of YBX1 with four predicted binding sites (BS) in the LDHA promoter sequence. G The LDHA promoter sequence was divided into 3 fragments containing the predicted BS, followed by primer design and ChIP-qPCR to verify YBX1 binding to the LDHA promoter region. H The LDHA promoter sequence was divided into 4 fragments, and the BS-1 site that was mainly bound was mutated, the luciferase-tagged truncated plasmids were designed. After co-transfection of the target and control Renilla plasmids into ccRCC cells, YBX1 binding to the LDHA promoter region was verified using dual-luciferase reporter assays. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: no significant difference. Unpaired two-sided Student’s t -test in A, G and H. Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Quantitative RT-PCR, Expressing, Knockdown, Over Expression, Binding Assay, Sequencing, ChIP-qPCR, Luciferase, Cotransfection, Control

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A Western blot analysis of LDHA protein expression levels in ACHN and 786-O cells after YBX1 knockdown and overexpression. B Dual-luciferase reporter assays evaluating the effects of YBX1 and LDHA on glycolysis-related signaling pathways in ccRCC cells co-transfected with target plasmids and control Renilla plasmids. C Western blot analysis of p-p65 (Ser536) and total p65 expression after overexpression of YBX1 and LDHA. D Immunohistochemical staining to detect protein expression of total p65 and p-p65 (Ser536) in tumor tissues from control and YBX1 knockdown groups in nude mice. Representative images and quantification. E, F Western blot ( E ) and qRT-PCR ( F ) analysis of LDHA knockdown efficiency in 786-O cells 72 h post-transfection with si- LDHA . G , H Western blot ( G ) and LDH activity ( H ) analysis of LDHA inhibition in 786-O cells treated with Oxamate (0-80 mM for 48 h). I , J Western blot analysis of p-p65 (Ser536) and total p65 in YBX1-overexpressing cells following LDHA knockdown ( I ) or Oxamate treatment (60 mM for 48 h) ( J ). K-N Lactate production ( K, L ) and cell proliferation ( M , N ) assays in YBX1-overexpressing cells after LDHA knockdown or Oxamate treatment. O Schematic diagram illustrating the YBX1-LDHA-NF-κB mechanism in ccRCC progression. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: no significant difference. Unpaired two-sided Student’s t -test in B , D , F and H . One-way ANOVA with correction for multiple comparisons in K and L. Two-way ANOVA with correction for multiple comparisons in M and N. Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Western Blot, Expressing, Knockdown, Over Expression, Luciferase, Protein-Protein interactions, Transfection, Control, Immunohistochemical staining, Staining, Quantitative RT-PCR, Activity Assay, Inhibition

Journal: Frontiers in Immunology

Article Title: Prognostic and immunological implications of cathepsin Z overexpression in prostate cancer

doi: 10.3389/fimmu.2025.1618487

Figure Lengend Snippet: Expression and prognostic significance of CTSZ across multiple cancer types. (A) Differential expression analysis of CTSZ in 26 cancer types demonstrating significantly elevated expression in tumor tissues compared to normal tissues. (B) Spatial transcriptomic analysis reveals that CTSZ expression is markedly higher in Mal than in Bdy and nMal regions. (C, D) Kaplan-Meier analysis showing that high CTSZ expression correlates with poorer OS and shorter PFS. (E) High CTSZ expression consistently associates with reduced OS and PFS across multiple cancer types. * p < 0.05. ** p < 0.01. *** p < 0.001.

Article Snippet: The antibodies used included rabbit anti-CTSZ monoclonal antibody (Cat. 16578-1-AP, Proteintech, USA), and rabbit anti-Tubulin polyclonal antibody (Cat. 10068-1-AP, Proteintech, USA).

Techniques: Expressing, Quantitative Proteomics

Journal: Frontiers in Immunology

Article Title: Prognostic and immunological implications of cathepsin Z overexpression in prostate cancer

doi: 10.3389/fimmu.2025.1618487

Figure Lengend Snippet: CTSZ expression and prognostic impact in PCa from TCGA-PRAD and MSKCC cohorts. (A–C) In the TCGA-PRAD cohort, CTSZ expression positively correlates with Gleason scores, T stage, and N stage. (D–F) In the MSKCC cohort, CTSZ expression positively associates with Gleason scores, T stage, and PSA levels. (G, H) Kaplan-Meier survival analysis indicates high CTSZ expression is linked with worse OS in both the TCGA-PRAD and MSKCC cohorts.

Article Snippet: The antibodies used included rabbit anti-CTSZ monoclonal antibody (Cat. 16578-1-AP, Proteintech, USA), and rabbit anti-Tubulin polyclonal antibody (Cat. 10068-1-AP, Proteintech, USA).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Prognostic and immunological implications of cathepsin Z overexpression in prostate cancer

doi: 10.3389/fimmu.2025.1618487

Figure Lengend Snippet: Validation of CTSZ expression and prognostic value in the AHMU-PC cohort and immunohistochemical analysis. (A) Higher CTSZ expression observed in PCa patients with Gleason scores of 8–10 compared to scores of 6–7. (B) Elevated CTSZ expression in PCa patients with extracapsular extension. (C) Increased CTSZ expression correlates with a biopsy-positive rate exceeding 50%. (D) Kaplan-Meier survival curve indicates significantly worse recurrence-free survival in patients with high CTSZ expression. (E) Representative immunohistochemical staining showing elevated CTSZ expression levels in PCa tissues.

Article Snippet: The antibodies used included rabbit anti-CTSZ monoclonal antibody (Cat. 16578-1-AP, Proteintech, USA), and rabbit anti-Tubulin polyclonal antibody (Cat. 10068-1-AP, Proteintech, USA).

Techniques: Biomarker Discovery, Expressing, Immunohistochemical staining, Staining

Journal: Frontiers in Immunology

Article Title: Prognostic and immunological implications of cathepsin Z overexpression in prostate cancer

doi: 10.3389/fimmu.2025.1618487

Figure Lengend Snippet: CTSZ expression in PCa cell lines and functional impact of CTSZ knockdown in PC3 and DU145 cells. (A) Comparative analysis of CTSZ expression in PCa cell lines versus normal prostate epithelial cells. (B) Validation of CTSZ knockdown efficiency in PC3 and DU145 cells by qPCR and WB analysis (C–F) CTSZ knockdown significantly suppressed cell proliferation (C) , colony formation (D) , invasion (E) , and migration (F) in both PC3 and DU145 cells. Data represent the mean ± standard deviation from three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: The antibodies used included rabbit anti-CTSZ monoclonal antibody (Cat. 16578-1-AP, Proteintech, USA), and rabbit anti-Tubulin polyclonal antibody (Cat. 10068-1-AP, Proteintech, USA).

Techniques: Expressing, Functional Assay, Knockdown, Biomarker Discovery, Migration, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Prognostic and immunological implications of cathepsin Z overexpression in prostate cancer

doi: 10.3389/fimmu.2025.1618487

Figure Lengend Snippet: Relationship between CTSZ expression and tumor immune infiltration in PCa. (A) Pathway enrichment analysis revealing activation of immune-related pathways in high vs. low CTSZ expression groups. (B) CTSZ expression positively correlates with immuneScore and stromalScore. (C, D) Positive correlation between CTSZ expression and infiltration of multiple immune cell types in PCa. (E) Comparison of immune cell infiltration profiles between high and low CTSZ expression subgroups. (F, G) CIBERSORT analysis confirming positive correlation between CTSZ expression and infiltration levels of total macrophages and M2 macrophages. All data were obtained from the TCGA-PRAD cohort. ns: not significant (p ≥ 0.05). * p < 0.05. ** p < 0.01. **** p < 0.0001.

Article Snippet: The antibodies used included rabbit anti-CTSZ monoclonal antibody (Cat. 16578-1-AP, Proteintech, USA), and rabbit anti-Tubulin polyclonal antibody (Cat. 10068-1-AP, Proteintech, USA).

Techniques: Expressing, Activation Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Prognostic and immunological implications of cathepsin Z overexpression in prostate cancer

doi: 10.3389/fimmu.2025.1618487

Figure Lengend Snippet: Spatial associations of CTSZ with immune checkpoint molecules and M2 macrophages in PCa tissues. (A) Immunohistochemistry results show a positive correlation between CTSZ and PD-1 expression in PCa tissues. (B) mIF images demonstrate spatial co-localization of CTSZ with immune checkpoint molecules PD-1 and PD-L1 in the tumor microenvironment. (C) mIF images reveal spatial proximity between CTSZ + tumor cells and CD163 + CD206 + M2 macrophages, suggesting a potential positive association. * p < 0.05. ** p < 0.01. *** p < 0.001.

Article Snippet: The antibodies used included rabbit anti-CTSZ monoclonal antibody (Cat. 16578-1-AP, Proteintech, USA), and rabbit anti-Tubulin polyclonal antibody (Cat. 10068-1-AP, Proteintech, USA).

Techniques: Immunohistochemistry, Expressing

Journal: Frontiers in Immunology

Article Title: Prognostic and immunological implications of cathepsin Z overexpression in prostate cancer

doi: 10.3389/fimmu.2025.1618487

Figure Lengend Snippet: Association between CTSZ expression and genetic mutations in PCa. (A) Elevated arm-level and focal amplification/deletion events observed in the high CTSZ expression subgroup. (B) Comparative analysis of gene mutation frequencies between CTSZ high- and low-expression groups. (C) Significantly higher CTSZ expression in TP53 -mutant PCa samples compared to TP53 wild-type. (D) TP53 signaling pathway enrichment identified in the CTSZ high-expression subgroup. (E) Kaplan-Meier analysis demonstrating worse prognosis in TP53-mutant PCa compared to wild-type cases. Genomic and transcriptomic data were retrieved from the TCGA-PRAD cohort.

Article Snippet: The antibodies used included rabbit anti-CTSZ monoclonal antibody (Cat. 16578-1-AP, Proteintech, USA), and rabbit anti-Tubulin polyclonal antibody (Cat. 10068-1-AP, Proteintech, USA).

Techniques: Expressing, Amplification, Mutagenesis

Journal: Frontiers in Immunology

Article Title: Prognostic and immunological implications of cathepsin Z overexpression in prostate cancer

doi: 10.3389/fimmu.2025.1618487

Figure Lengend Snippet: KEGG enrichment analysis linked to CTSZ expression. (A–C) KEGG enrichment analysis identified CAM, VEGF, and STAT signaling pathways as significantly enriched in association with high CTSZ expression. Enrichment analysis was based on gene expression data from the TCGA-PRAD cohort.

Article Snippet: The antibodies used included rabbit anti-CTSZ monoclonal antibody (Cat. 16578-1-AP, Proteintech, USA), and rabbit anti-Tubulin polyclonal antibody (Cat. 10068-1-AP, Proteintech, USA).

Techniques: Expressing, Protein-Protein interactions, Gene Expression

Journal: Cell Communication and Signaling : CCS

Article Title: Primary cilium and TULP3-dependent ciliary targeting of ACE2 in SARS-CoV-2 tropism

doi: 10.1186/s12964-025-02519-y

Figure Lengend Snippet: Dysregulated expression of primary cilium-associated genes in SARS-CoV-2 infected patient lung samples. A The Venn diagram presents genome-wide RNA sequencing results from patient lung samples, revealing differentially expressed genes (DEGs) associated with primary cilium. The DEGs were defined by log2 fold change > 1.5, with adjusted P < 0.05. P values were adjusted using the Benjamini–Hochberg correction. The analysis showing 3,972 DEGs in patient samples and 956 primary cilia-related genes in CiliaCarta, with 129 overlapping genes functionally linked to primary cilium. B The list of 129 ciliary DEGs identified in the SARS-CoV-2 patient lung samples. Gene ranked by the differently expressed level. Blue dots represent down-regulated genes, red dots represent up-regulated genes. Colour gradient and size of dots correlate with the degree of fold change. C Volcano plots of 129 DEGs in the patient lung samples overlap with the genes associated with ciliary functions. DEGs at -log10 > 2 and |log2 fold change|> 2 were indicated. (adjusted p values < 0.05, P values adjusted using the Benjamini–Hochberg correction) 61 up-regulated genes presented as red dots, 68 down-regulated genes presented as blue dots. Top 15 and two prominent ciliary genes ARL13B and ADCY3 are indicated. D Gene Ontology (GO) enrichment analysis for Biological Processes among ciliary DEGs from SARS-CoV-2 patient lung samples. Terms are ranked by their enrichment score, calculated using Gene Set Enrichment Analysis (GSEA). E Gene Set Enrichment Analysis (GSEA) reveals key signaling pathways associated with ciliary DEGs from SARS-CoV-2 patient lungs, ranked by enrichment score. F Ciliary DEGs from SARS-CoV-2 patient lung samples grouped by ciliary functional category. G Venn diagram and table show overlap between ACE2 interactors from BioGRID (979 proteins) and ciliary genes from CiliaCarta (956 genes) and Syscilia (303 genes). 56 and 14 overlapping genes were identified in each cilia database, respectively. Two well-studied ciliary components, namely ARL13B and TULP3 are identified in the intersections (highlighted yellow). H Gene Ontology (GO) enrichment analysis for Biological Processes among ciliary ACE2 interactors. Terms are ranked by their enrichment score, calculated using Gene Set Enrichment Analysis (GSEA)

Article Snippet: The primary antibodies used include, anti-ACE2 antibody (1:3000, cat. number: AF933, R&D systems), anti-ARL13B antibody (1:3000, cat. number: 17711–1-AP, Proteintech), anti-IFT88 antibody (1:2000, cat. number: ab42497, Abcam), anti-TULP3 antibody (1:500, cat. number: 13637–1-AP, Proteintech), anti-KIF3A antibody (1:4000, cat. number: ab11259, Abcam), anti-NRP1 antibody (1:1000, cat. number: ab81321, Abcam), anti-GAPDH antibody (1:8000, cat. number: ab8245, Abcam), anti-ß-ACTIN antibody (1:2000, cat. number: sc-47778, Santa Cruz Biotechnology).

Techniques: Expressing, Infection, Genome Wide, RNA Sequencing, Protein-Protein interactions, Functional Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Primary cilium and TULP3-dependent ciliary targeting of ACE2 in SARS-CoV-2 tropism

doi: 10.1186/s12964-025-02519-y

Figure Lengend Snippet: TULP3 colocalizes with ACE2 on the primary cilium axoneme and physically interacts with ACE2. A Representative immunofluorescence images show the accumulation and co-localization of TULP3 and ACE2 on the primary cilium of A549 cells. B Histogram illustrates co-localization profile of endogenous ACE2 and TULP3 expressions on ARL13B-positive cilia axoneme of A549. C Mander’s Coefficients analysis of ACE2 and TULP3 co-localization in cytoplasm versus primary cilia of A549 cell. The data represents quantifications from three independent experiments, with 10–20 data points measured in each experiment. Statistical analysis is performed using Paired Student’s t-Test, Two tailed. P values: ** < 0.01, *** < 0.001, **** < 0.0001. D Representative Western-blot images illustrate the co-IP experiment of ACE2 and TULP3 on ACE2-GFP overexpressing HEK293T cell line. IP: immunoprecipitation; IB: immunoblot. E Graph depicts densitometry analysis of TULP3 signal intensity in ACE2 and IgG immunoprecipitation groups. The data represents quantifications from three independent experiments. Statistical analysis is performed using Unpaired Student’s t-Test, Two tailed. P value * < 0.05. F Graph depicts densitometry analysis of ACE2 signal intensity in TULP3 and IgG immunoprecipitation groups. The data represents quantifications from three independent experiments. Statistical analysis is performed using Unpaired Student’s t-Test, Two tailed. P value * < 0.05. G Representative Western-blot images illustrate the co-IP experiment of ACE2 and ARL13B on ACE2-GFP overexpressing HEK293T cell line. IP: immunoprecipitation; IB: immunoblot. H TULP3–ACE2 interaction interface predicted from AlphaFold3 . TULP3 (light blue, residues 271–430) and ACE2 (light red, residues 749–773) are shown with key interface residues highlighted as sticks (blue for TULP3, red for ACE2). The TULP3 cargo-binding face (β8–β12, highlighted in marine) engages the membrane-proximal ACE2 segment. Yellow dashed lines represent top recurring contacts across models. ACE2 residue I761, involved in all major contacts, is labelled

Article Snippet: The primary antibodies used include, anti-ACE2 antibody (1:3000, cat. number: AF933, R&D systems), anti-ARL13B antibody (1:3000, cat. number: 17711–1-AP, Proteintech), anti-IFT88 antibody (1:2000, cat. number: ab42497, Abcam), anti-TULP3 antibody (1:500, cat. number: 13637–1-AP, Proteintech), anti-KIF3A antibody (1:4000, cat. number: ab11259, Abcam), anti-NRP1 antibody (1:1000, cat. number: ab81321, Abcam), anti-GAPDH antibody (1:8000, cat. number: ab8245, Abcam), anti-ß-ACTIN antibody (1:2000, cat. number: sc-47778, Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Two Tailed Test, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Binding Assay, Membrane, Residue

Journal: Cell Communication and Signaling : CCS

Article Title: Primary cilium and TULP3-dependent ciliary targeting of ACE2 in SARS-CoV-2 tropism

doi: 10.1186/s12964-025-02519-y

Figure Lengend Snippet: TULP3 regulates ACE2 localization to the primary cilium. A Representative immunofluorescence images showing reduced ACE2 expression on the primary cilium in the shControl and TULP3 KD groups. B Graphs showing reduced ACE2 enrichment ratio in the (top) TULP3 and (bottom) ARL13B KD groups. ACE2 enrichment ratio was calculated as (ACE2 intensity per area on primary cilium)/(ACE2 intensity per area on whole cell). The data represents quantifications from three to four independent experiments, n = 14–17 measurements in each experiment. Statistical analysis is performed using (top) One-way ANOVA followed by Dunnett’s multiple comparisons and (bottom) Unpaired Student’s t-test. P values: * < 0.05, *** < 0.001, ns = not significant. C Representative immunofluorescence images show ciliary ACE2 expression on the primary cilium in TULP3 WT and TULP3 mut12 overexpression groups (pseudo-color magenta). D Graph shows ciliary ACE2 enrichment ratio in the TULP3 WT rescue and TULP3. mut12 overexpression groups. ACE2 enrichment ratio was calculated as (ACE2 intensity per area on primary cilium)/(ACE2 intensity per area on whole cell). The data represents quantifications from four independent experiments, n = 10–20 measurements in each experiment. Statistical analysis is performed using One-way ANOVA followed by Tukey’s multiple comparisons test. P values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001

Article Snippet: The primary antibodies used include, anti-ACE2 antibody (1:3000, cat. number: AF933, R&D systems), anti-ARL13B antibody (1:3000, cat. number: 17711–1-AP, Proteintech), anti-IFT88 antibody (1:2000, cat. number: ab42497, Abcam), anti-TULP3 antibody (1:500, cat. number: 13637–1-AP, Proteintech), anti-KIF3A antibody (1:4000, cat. number: ab11259, Abcam), anti-NRP1 antibody (1:1000, cat. number: ab81321, Abcam), anti-GAPDH antibody (1:8000, cat. number: ab8245, Abcam), anti-ß-ACTIN antibody (1:2000, cat. number: sc-47778, Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Expressing, Over Expression

Journal: Cell Communication and Signaling : CCS

Article Title: Primary cilium and TULP3-dependent ciliary targeting of ACE2 in SARS-CoV-2 tropism

doi: 10.1186/s12964-025-02519-y

Figure Lengend Snippet: Schematic diagram illustrating our proposed model. TULP3 functions as a ciliary adaptor protein that governs the targeting of ACE2 to the ciliary axoneme, acting partially through anchoring to the IFT-A transport complex

Article Snippet: The primary antibodies used include, anti-ACE2 antibody (1:3000, cat. number: AF933, R&D systems), anti-ARL13B antibody (1:3000, cat. number: 17711–1-AP, Proteintech), anti-IFT88 antibody (1:2000, cat. number: ab42497, Abcam), anti-TULP3 antibody (1:500, cat. number: 13637–1-AP, Proteintech), anti-KIF3A antibody (1:4000, cat. number: ab11259, Abcam), anti-NRP1 antibody (1:1000, cat. number: ab81321, Abcam), anti-GAPDH antibody (1:8000, cat. number: ab8245, Abcam), anti-ß-ACTIN antibody (1:2000, cat. number: sc-47778, Santa Cruz Biotechnology).

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: Primary cilium and TULP3-dependent ciliary targeting of ACE2 in SARS-CoV-2 tropism

doi: 10.1186/s12964-025-02519-y

Figure Lengend Snippet: Attachment and accumulation of the viral spike protein on the ACE2-enriched primary cilium. A Representative immunofluorescence images show the negative control without exposure to spike protein. A ” Histogram illustrating the ciliary co-localization and expression profiles on of spike and ACE2 signals the negative control. B Representative immunofluorescence images show the accumulation and co-localization of viral recombinant spike protein on endogenously expressed ACE2 on the ARL13B. + primary cilium. B ” Histogram illustrating the co-localization and expression profile of three fluorescence channels reflecting viral spike protein, ARL13B and ACE2 respectively, on the primary cilium. C Graph depicts the spike expression intensity on ACE2-negative and ACE2-positive primary cilia. ACE2 pixel intensities per cilia area greater than 500 were defined as ACE2-positive primary cilia, whereas below 500 were counted as ACE2-negative primary cilia. The data represent quantifications from three independent experiments, with n = 10–20 for each group in each experiment. P values: ** < 0.01

Article Snippet: The primary antibodies used include, anti-ACE2 antibody (1:3000, cat. number: AF933, R&D systems), anti-ARL13B antibody (1:3000, cat. number: 17711–1-AP, Proteintech), anti-IFT88 antibody (1:2000, cat. number: ab42497, Abcam), anti-TULP3 antibody (1:500, cat. number: 13637–1-AP, Proteintech), anti-KIF3A antibody (1:4000, cat. number: ab11259, Abcam), anti-NRP1 antibody (1:1000, cat. number: ab81321, Abcam), anti-GAPDH antibody (1:8000, cat. number: ab8245, Abcam), anti-ß-ACTIN antibody (1:2000, cat. number: sc-47778, Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Negative Control, Expressing, Recombinant, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Primary cilium and TULP3-dependent ciliary targeting of ACE2 in SARS-CoV-2 tropism

doi: 10.1186/s12964-025-02519-y

Figure Lengend Snippet: Host cell entry factors for SARS-CoV-2 are endogenously expressed and enriched in primary cilia across multiple SARS-CoV-2 susceptible cell types. A Representative immunofluorescence images show the endogenous expression of ACE2 (green) on the primary cilium (red) of human lung carcinoma, A549, human retinal pigment epithelial cells (hTERT-RPE1), as well as neuronal cell types namely neuroblastoma, SH-SY5Y, and human induced-pluripotent stem cell derived neural progenitor cells (hiPSC-NPCs) respectively. B The violin plots depict the quantification of the per area expression intensity of ACE2 in the primary cilium in relation to the entire cell. The data represents quantifications from three independent experiments. Statistical analysis is performed using Wilcoxon matched-pairs signed rank test. P values: ** < 0.01,**** < 0.0001. ~ 10 to 25 cells were analysed in each group of three independent experiments. C The violin plot depicts the percentage of ACE2-expressing primary cilia across different human cell types that are susceptible to SARS-CoV-2 infection. Data illustrates three to eight independent experiments for each group. Statistical analysis is performed using Kruskal–Wallis Test and Dunn’s multiple comparisons test. P values: * < 0.05, ** < 0.01. D Representative immunofluorescence images showing the endogenous expression of NRP1 (green) on the primary cilium (red) of human lung carcinoma, A549 and hiPSC-NPCs

Article Snippet: The primary antibodies used include, anti-ACE2 antibody (1:3000, cat. number: AF933, R&D systems), anti-ARL13B antibody (1:3000, cat. number: 17711–1-AP, Proteintech), anti-IFT88 antibody (1:2000, cat. number: ab42497, Abcam), anti-TULP3 antibody (1:500, cat. number: 13637–1-AP, Proteintech), anti-KIF3A antibody (1:4000, cat. number: ab11259, Abcam), anti-NRP1 antibody (1:1000, cat. number: ab81321, Abcam), anti-GAPDH antibody (1:8000, cat. number: ab8245, Abcam), anti-ß-ACTIN antibody (1:2000, cat. number: sc-47778, Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Expressing, Derivative Assay, Infection

Journal: Cell Communication and Signaling : CCS

Article Title: Primary cilium and TULP3-dependent ciliary targeting of ACE2 in SARS-CoV-2 tropism

doi: 10.1186/s12964-025-02519-y

Figure Lengend Snippet: Genetic perturbation of primary cilia formation reduces SARS-CoV-2 infection in human host cells. A - B Representative Western blot images and the corresponding graphs show the knockdown efficiency of sh ARL13B and sh IFT88 in the ( A ) A549 and ( B ) RPE1 knockdown cells. Knockdown efficiency was quantified by normalizing the signal intensity of the target protein from each knockdown sample to its respective control group. The data represents quantifications from three independent experiments. Statistical analysis is performed using One-way ANOVA followed by Dunnett’s multiple comparisons test. P values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. ns = not significant. Error bar: SD. C Representative immunofluorescence images show a reduced number of ciliated cells in the ARL13B KD and IFT88 KD groups in A549 (top panel) and RPE1 (bottom panel) cell lines. D - E Graphs depict the percentage of primary cilia-bearing (ciliated) cells in the ARL13B KD and IFT88 KD groups in ( D ) A549 and ( E ) RPE1 respectively. The data represents quantifications from five independent experiments. Statistical analysis is performed using One-way ANOVA followed by Dunnett’s multiple comparisons test. P values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. ns = not significant. F - G Graphs depict flow cytometry analysis of the relative infection rate of different SARS-CoV-2-GFP pseudovirus variants upon perturbation of primary cilia in the ARL13B - and IFT88- knockdown human lung and retinal cells respectively. The percentage of infected cells was determined by flow cytometry analysis of GFP-positive infected cells. The data represents quantifications from three independent experiments. Each dot represents the mean value of an independent experiment. Statistical analysis is performed using Two-way ANOVA, Tukey's multiple comparisons test. P values: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. ns = not significant. Error bar: S.E.M. H Schematic diagram summarizes the findings, showing that the loss of ACE2-enriched cilia decreases SARS-CoV-2 viral uptake. Created with https://BioRender.com

Article Snippet: The primary antibodies used include, anti-ACE2 antibody (1:3000, cat. number: AF933, R&D systems), anti-ARL13B antibody (1:3000, cat. number: 17711–1-AP, Proteintech), anti-IFT88 antibody (1:2000, cat. number: ab42497, Abcam), anti-TULP3 antibody (1:500, cat. number: 13637–1-AP, Proteintech), anti-KIF3A antibody (1:4000, cat. number: ab11259, Abcam), anti-NRP1 antibody (1:1000, cat. number: ab81321, Abcam), anti-GAPDH antibody (1:8000, cat. number: ab8245, Abcam), anti-ß-ACTIN antibody (1:2000, cat. number: sc-47778, Santa Cruz Biotechnology).

Techniques: Infection, Western Blot, Knockdown, Control, Immunofluorescence, Flow Cytometry

Journal: Acta Pharmacologica Sinica

Article Title: Paeoniflorin-6′-O-benzene sulfonate alleviates collagen-induced arthritis in mice by downregulating BAFF-TRAF2-NF-κB signaling: comparison with biological agents

doi: 10.1038/s41401-018-0169-5

Figure Lengend Snippet: The effect of CP-25 on the expressions of CD19+TRAF2+ cells of mice. a The effect of CP-25 on the expressions of CD19+TRAF2+ cells of mice by flow cytometry. b The percentages of CD19+TRAF2+ cells were observed and the effect of CP-25. The expressions of CD19+TRAF2+ cells were increased after BAFF induction CP-25, rituximab, and etanercept reduced the expressions of CD19+TRAF2+ cells stimulated by BAFF. **P < 0.01 vs. Control. ##P < 0.01 vs. BAFF. $P < 0.05 vs. CP-25

Article Snippet: The Brilliant Violet 421 anti-mouse CD19 antibody was purchased from Biolegend Co., Ltd., and the TRAF2 (H-10) Alexa Fluor 647 antibody was purchased from Santa Cruz Biotechnology Inc.

Techniques: Flow Cytometry, Control

Journal: Acta Pharmacologica Sinica

Article Title: Paeoniflorin-6′-O-benzene sulfonate alleviates collagen-induced arthritis in mice by downregulating BAFF-TRAF2-NF-κB signaling: comparison with biological agents

doi: 10.1038/s41401-018-0169-5

Figure Lengend Snippet: The overexpression of TRAF2 in HEK 293 cell and the effect of CP-25. a The figure showed transfection of PIRES2-EGFP-TRAF2 plasmid and the effect of CP-25. b The overexpression of TRAF2 was analyzed by Western blotting. c The effect of CP-25 on the expressions of TRAF2. **P < 0.01 vs. Control. ##P < 0.01 vs. PIRES2-EGFP. &P < 0.05 vs. PIRES2-EGFP-TRAF2

Article Snippet: The Brilliant Violet 421 anti-mouse CD19 antibody was purchased from Biolegend Co., Ltd., and the TRAF2 (H-10) Alexa Fluor 647 antibody was purchased from Santa Cruz Biotechnology Inc.

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, Control

Journal: Acta Pharmacologica Sinica

Article Title: Paeoniflorin-6′-O-benzene sulfonate alleviates collagen-induced arthritis in mice by downregulating BAFF-TRAF2-NF-κB signaling: comparison with biological agents

doi: 10.1038/s41401-018-0169-5

Figure Lengend Snippet: The effect of CP-25 on the NF-κB signaling molecules expressions. a The expression of TRAF2, MKK3, MKK6, p-P38, and p-NF-κB65 in B cells of mice were analyzed by Western blotting. b The effect of CP-25 on the expressions of key molecules in NF-κB signaling. **P < 0.01 vs. Normal. #P < 0.05, ##P < 0.01 vs. CIA. $P < 0.05, $$P < 0.01 vs. CP-25

Article Snippet: The Brilliant Violet 421 anti-mouse CD19 antibody was purchased from Biolegend Co., Ltd., and the TRAF2 (H-10) Alexa Fluor 647 antibody was purchased from Santa Cruz Biotechnology Inc.

Techniques: Expressing, Western Blot

Journal: Acta Pharmacologica Sinica

Article Title: Paeoniflorin-6′-O-benzene sulfonate alleviates collagen-induced arthritis in mice by downregulating BAFF-TRAF2-NF-κB signaling: comparison with biological agents

doi: 10.1038/s41401-018-0169-5

Figure Lengend Snippet: To summary the effect of CP-25, rituximab and etanercept on BAFF/BAFF receptor-TRAF2-NF-κB signaling in B cell on CIA mice. The whole criteria of CIA mice were increased, the function of B cells was abnormal, and NF-κB signaling pathways mediated by BAFF in B cells of CIA mice were activated. CP-25 reduced the whole criteria, T/B cells proliferations, and downregulated abnormal B-cell function and the key molecules of BAFF-NF-κB signaling pathways. The action intensity of rituximab and etanercept on B cells was strong, which might be one of mechanisms of adverse reaction. AI arthritis index, BAFF B-cell activating factor, BAFFR B-cell activating factor receptor, CIA collagen-induced arthritis, CREB cAMP response element binding protein, MKK3 mitogen-activated protein kinase kinase 3, MKK6 mitogen-activated protein kinase kinase 6, p-P38 phosphorylated p38 mitogen-activated protein kinase, NF-κB nuclear factor of kappa B, IκB inhibit of nuclear factor of kappa B, SJC swollen joint counts, TACI transmembrane activator and calcium modulator and cyclophilin ligand interactor, TRAF2 tumor necrosis factor receptor-associated factors 2

Article Snippet: The Brilliant Violet 421 anti-mouse CD19 antibody was purchased from Biolegend Co., Ltd., and the TRAF2 (H-10) Alexa Fluor 647 antibody was purchased from Santa Cruz Biotechnology Inc.

Techniques: Protein-Protein interactions, Cell Function Assay, Binding Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: A common variant of RIP3 promoter region is associated with poor prognosis in heart failure patients by influencing SOX17 binding

doi: 10.1111/jcmm.14408

Figure Lengend Snippet: rs3212247 is responsible for haplotype‐specific difference in transcriptional activity. (A), Map of single‐nucleotide polymorphisms (SNPs) in the promoter and 5'UTR of Receptor‐interacting protein kinase 3 genotyped in 200 healthy individuals. The eight SNPs (indicated with boxes), which were predicted to be possible functional at RegulomeDB, comprise a single haplotype and define the 1.8 and 0.2 kb in promoter and 5'UTR, respectively. (B), Firefly luciferase expression from constructs transfected into AC16. Both the major (Hap1) and minor (Hap2) haplotypes of the 1.8 kb in promoter region were subcloned into PGL3‐basic vector. (C‐I), Luciferase assays for rs3212247, rs3834521, rs3759625, rs3212249, rs3759630, rs3212246 rs3212250‐51 (rs3212250 and rs3212251 were combined as a haplotype as they are close enough) were conducted using AC16. Approximately 250‐bp regions encompassing corresponding variants were cloned into PGL3‐basic vector. Luciferase activity was measured 48 h after transfection and was normalized against Renilla luciferase activity. Values are mean ± SE of three independent experiments each corresponding to at least six replicates. ** P < 0.01, **** P < 0.0001, NS, not significant

Article Snippet: Total plasma RIP3 levels were analysed using a Human RIP3 ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer's instructions.

Techniques: Activity Assay, Functional Assay, Luciferase, Expressing, Construct, Transfection, Plasmid Preparation, Clone Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: A common variant of RIP3 promoter region is associated with poor prognosis in heart failure patients by influencing SOX17 binding

doi: 10.1111/jcmm.14408

Figure Lengend Snippet: Characteristics of RIP3 variants identified by Sanger sequencing

Article Snippet: Total plasma RIP3 levels were analysed using a Human RIP3 ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer's instructions.

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: A common variant of RIP3 promoter region is associated with poor prognosis in heart failure patients by influencing SOX17 binding

doi: 10.1111/jcmm.14408

Figure Lengend Snippet: SOX17 binds to the rs3212247‐T allele and mediates allelic expression. (A), Firefly luciferase assays were performed with 250‐bp rs3212247‐T or ‐C allele constructs cotransfected into AC16 cells with SOX17 vector encoding sox17. Luciferase activity was measured 48 h after transfection and was normalized against Renilla luciferase activity. (B), AC16 cells were transfected with pcDNA3.1 and SOX17 respectively. Protein was harvested 48 h after transfection and Receptor‐interacting protein kinase 3 (RIP3) expression was normalized against GAPDH. * P < 0.05

Article Snippet: Total plasma RIP3 levels were analysed using a Human RIP3 ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer's instructions.

Techniques: Expressing, Luciferase, Construct, Plasmid Preparation, Activity Assay, Transfection

Journal: Journal of Cellular and Molecular Medicine

Article Title: A common variant of RIP3 promoter region is associated with poor prognosis in heart failure patients by influencing SOX17 binding

doi: 10.1111/jcmm.14408

Figure Lengend Snippet: In vivo plasma concentrations of Receptor‐interacting protein kinase 3 (RIP3) in control group (A) HF NYHA class II group (B) HF NYHA class III group (C) HF NYHA class IV group (D) and among these four groups (E). The data are presented as box (25th percentile, median and 75th percentile)

Article Snippet: Total plasma RIP3 levels were analysed using a Human RIP3 ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer's instructions.

Techniques: In Vivo, Clinical Proteomics, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: A common variant of RIP3 promoter region is associated with poor prognosis in heart failure patients by influencing SOX17 binding

doi: 10.1111/jcmm.14408

Figure Lengend Snippet: Association between RIP3 variants and heart failure

Article Snippet: Total plasma RIP3 levels were analysed using a Human RIP3 ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer's instructions.

Techniques: Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: A common variant of RIP3 promoter region is associated with poor prognosis in heart failure patients by influencing SOX17 binding

doi: 10.1111/jcmm.14408

Figure Lengend Snippet: Association of the RIP3 polymorphisms with cardiac mortality or transplantation in chronic heart failure

Article Snippet: Total plasma RIP3 levels were analysed using a Human RIP3 ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer's instructions.

Techniques: Transplantation Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: A common variant of RIP3 promoter region is associated with poor prognosis in heart failure patients by influencing SOX17 binding

doi: 10.1111/jcmm.14408

Figure Lengend Snippet: Effect of SOX17 on Regulation of Receptor‐interacting protein kinase 3 (RIP3) Transcription. The variant rs3212247 in RIP3 promoter region is a T‐ to‐C change and is predicted to locate in the binding site of SOX17. Rs3212247‐C allele destroys the SOX17 binding site in the promoter region of RIP3 and subsequently results in increased transcription and translation of RIP3, followed by increased necroptosis of cardiomyocyte under various external stimuli, which leads to poor prognosis of heart failure (HF)

Article Snippet: Total plasma RIP3 levels were analysed using a Human RIP3 ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer's instructions.

Techniques: Variant Assay, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Interactive Roles of Ets-1, Sp1, and Acetylated Histones in the Retinoic Acid-dependent Activation of Guanylyl Cyclase/Atrial Natriuretic Peptide Receptor-A Gene Transcription

doi: 10.1074/jbc.m110.132795

Figure Lengend Snippet: FIGURE 2. Effect of overexpression and knockdown of RAR and RXR on ATRA-induced Npr1 gene transcription. A, luciferase activity of the Npr1 promoter construct 356/55 cotransfected with RAR or/and RXR expression plasmid (0.5 g) and stimulated with ATRA (0.5 M) or 0.1% dimethyl sulfoxide (vehicle control). B, representative protein expression lev- els of RAR and RXR in transfected ATRA-stimulated cells as analyzed by Western blot and -actin taken as loading control. C, luciferase activity of the Npr1 promoter cotransfected with RAR and/or RXR siRNA followed by stimulation with ATRA (0.5 M). D, Western blot analysis of siRNA-mediated knockdownofendogenousRARandRXRintransfectedcellsand-actinas loading control. E, luciferase activity of the Npr1 promoter in cells pretreated with increasing concentrations of Ro 41–5253 for 1 h and stimulated with ATRA. F, Npr1 mRNA levels in cells pretreated with Ro 41–5253 and stimulated with ATRA as determined by real-time RT-PCR. Bars indicate the fold induc- tion of Npr1 mRNA levels relative to untreated controls with -actin as inter- nal control. Data represent the mean S.E. of four independent experiments in triplicate. The downward arrow indicates percent repression; UT, untrans- fected; WB, Western blot; Ctrl, control. **, p 0.01; ***, p 0.001.

Article Snippet: Antibodies anti-Ets-1 (N-276), anti-Sp1 (H-225), anti-RAR (C-20), anti-RXR ( N-197), Ac-histone H4 (K-8), histone H3 (FL-136), anti-rabbit IgG-FITC, protein A-agarose, Ets-1 siRNA (mouse), Sp1 siRNA (mouse), RAR siRNA (mouse), RXR siRNA (mouse), and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Over Expression, Knockdown, Luciferase, Activity Assay, Construct, Expressing, Plasmid Preparation, Control, Transfection, Western Blot, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: Interactive Roles of Ets-1, Sp1, and Acetylated Histones in the Retinoic Acid-dependent Activation of Guanylyl Cyclase/Atrial Natriuretic Peptide Receptor-A Gene Transcription

doi: 10.1074/jbc.m110.132795

Figure Lengend Snippet: FIGURE3.CombinedeffectofEts-1andATRAonNpr1genetranscription.A,aschematicmapofNpr1promoter region 356 to 55 showing position and sequence of Sp1 and Ets-1 binding sites. B, upper panel shows a sche- matic map of the Npr1 promoter region 356 to 55 containing Sp1 and Ets-1 binding sites and mutant Ets-1 (Ets mut)sites.LuciferaseactivityoftheNpr1promotercontainingwild-typeormutantEts-1bindingsitesintransfected and ATRA-stimulated cells. C, Western blot analysis of Ets-1 in transfected cells with -actin expression as loading control. Bars represent the densitometry quantification of Ets-1 protein in Ets-1-transfected and/or ATRA-treated cellscomparedwithcontrolcells.D,luciferaseactivityoftheNpr1promotercotransfectedwithEts-1orcontrolsiRNA andstimulatedwithATRA.E,WesternblotanalysisofknockdowneffectofEts-1siRNAonendogenousEts-1protein in transfected cells and -actin as loading control. Data shown represent the mean S.E. of three independent experiments in triplicates. An upward arrow indicates fold induction; a downward arrow indicates percent repres- sion; UT, untransfected; WB, Western blot; Ctrl, control. **, p 0.01; ***, p 0.001.

Article Snippet: Antibodies anti-Ets-1 (N-276), anti-Sp1 (H-225), anti-RAR (C-20), anti-RXR ( N-197), Ac-histone H4 (K-8), histone H3 (FL-136), anti-rabbit IgG-FITC, protein A-agarose, Ets-1 siRNA (mouse), Sp1 siRNA (mouse), RAR siRNA (mouse), RXR siRNA (mouse), and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Sequencing, Binding Assay, Mutagenesis, Western Blot, Transfection, Expressing, Control

Journal: Journal of Biological Chemistry

Article Title: Interactive Roles of Ets-1, Sp1, and Acetylated Histones in the Retinoic Acid-dependent Activation of Guanylyl Cyclase/Atrial Natriuretic Peptide Receptor-A Gene Transcription

doi: 10.1074/jbc.m110.132795

Figure Lengend Snippet: FIGURE 5. Role of Sp1 in ATRA-mediated Npr1 gene transcription. A, lucif- erase activity of the Npr1 promoter construct 356/55 cotransfected with Sp1 expression plasmid and treated with ATRA. B, Western blot analysis of Sp1 protein expression in transfected cells, and -actin expression is shown as loading control. Bars represent the densitometry quantification of Sp1 pro- tein in Sp1-transfected or/and ATRA-treated cells compared with control cells. C, quantitative ChIP assay demonstrating recruitment of Sp1 to the Npr1 promoter in presence and absence of ATRA. DNA purified from the immuno- precipitated chromatin was amplified by real-time PCR. The upper panel shows a representative PCR gel picture. D, Western blot analysis of Sp1 siRNA-mediated knockdown of endogenous Sp1 in transfected cells and -actin as loading control. Bars show luciferase activity of the Npr1 promoter cotransfected with Sp1 siRNA and stimulated with ATRA. E, luciferase activity of the Npr1 promoter in cells pretreated with mithramycin A and induced with ATRA. An upward arrow indicates fold induction; a downward arrow indi- cates percent repression. Data shown represent the mean S.E. of three independent experiments in triplicates. Ab, antibody; WB, Western blot; Ctrl, control; Mith A, mithramycin A. *, p 0.05; **, p 0.01; ***, p 0.001.

Article Snippet: Antibodies anti-Ets-1 (N-276), anti-Sp1 (H-225), anti-RAR (C-20), anti-RXR ( N-197), Ac-histone H4 (K-8), histone H3 (FL-136), anti-rabbit IgG-FITC, protein A-agarose, Ets-1 siRNA (mouse), Sp1 siRNA (mouse), RAR siRNA (mouse), RXR siRNA (mouse), and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activity Assay, Construct, Expressing, Plasmid Preparation, Western Blot, Transfection, Control, Purification, Amplification, Real-time Polymerase Chain Reaction, Knockdown, Luciferase

Journal: Journal of Biological Chemistry

Article Title: Interactive Roles of Ets-1, Sp1, and Acetylated Histones in the Retinoic Acid-dependent Activation of Guanylyl Cyclase/Atrial Natriuretic Peptide Receptor-A Gene Transcription

doi: 10.1074/jbc.m110.132795

Figure Lengend Snippet: FIGURE 6. Interaction of Ets-1 and Sp1 in RAR/RXR-mediated ATRA effect on Npr1 transcription. A, luciferase activity of the Npr1 promoter construct 356/55 cotransfected with RAR or/and RXR expression plasmids and Ets-1 and/or Sp1 siRNAs and treated with ATRA (0.5 M). Sequential ChIP assays demonstrating in vivo recruitment of RAR to the Npr1 promoter by Ets-1 (B) and Sp1 (C) in untreated (upper panel) and ATRA-stimulated (0.5 M) (middle panel) cells. Direct binding of RAR in ATRA-treated and untreated cellsisshowninthelowerpanel.TheintensityofDNAbandswasquantifiedby Alpha Innotech analysis software. Representative DNA gels from four inde- pendent experiments are shown. Data represent the mean S.E. of four independent experiments in triplicate. Ab, antibody. ***, p 0.001.

Article Snippet: Antibodies anti-Ets-1 (N-276), anti-Sp1 (H-225), anti-RAR (C-20), anti-RXR ( N-197), Ac-histone H4 (K-8), histone H3 (FL-136), anti-rabbit IgG-FITC, protein A-agarose, Ets-1 siRNA (mouse), Sp1 siRNA (mouse), RAR siRNA (mouse), RXR siRNA (mouse), and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Luciferase, Activity Assay, Construct, Expressing, In Vivo, Binding Assay, Software

Journal: Journal of Biological Chemistry

Article Title: Interactive Roles of Ets-1, Sp1, and Acetylated Histones in the Retinoic Acid-dependent Activation of Guanylyl Cyclase/Atrial Natriuretic Peptide Receptor-A Gene Transcription

doi: 10.1074/jbc.m110.132795

Figure Lengend Snippet: FIGURE8.Schematicpresentationofproposedinteractionsbetweenreg- ulatory elements during ATRA-mediated regulation of the Npr1 gene. The proposed diagram indicates that ATRA increases Npr1 gene transcription and expression in association of Ets-1, Sp1, histone acetylation. ATRA induces Ets-1 protein expression via its receptor RAR and enhances Ets-1 and Sp1 binding to the Npr1 promoter. In the absence of functional RARE, RAR asso- ciates with the Ets-1Sp1 complex on the Npr1 promoter and mediates ATRA effects. Furthermore, ATRA acetylates histone H3 and H4 around the Ets-1 and Sp1 binding sites in the Npr1 promoter. Histone acetylation promotes localized unwinding of DNA and allows transcription factors to bind in the region. RA, retinoic acid. The closed upward arrow indicates an increased Ets-1 protein expression; the open upward arrow indicates an increased binding of Ets-1Sp1RAR complex to the Npr1 gene promoter.

Article Snippet: Antibodies anti-Ets-1 (N-276), anti-Sp1 (H-225), anti-RAR (C-20), anti-RXR ( N-197), Ac-histone H4 (K-8), histone H3 (FL-136), anti-rabbit IgG-FITC, protein A-agarose, Ets-1 siRNA (mouse), Sp1 siRNA (mouse), RAR siRNA (mouse), RXR siRNA (mouse), and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Binding Assay, Functional Assay

Journal: Journal of Biological Chemistry

Article Title: Interactive Roles of Ets-1, Sp1, and Acetylated Histones in the Retinoic Acid-dependent Activation of Guanylyl Cyclase/Atrial Natriuretic Peptide Receptor-A Gene Transcription

doi: 10.1074/jbc.m110.132795

Figure Lengend Snippet: FIGURE 7. Effect of ATRA on the association of acetylated histones H3 and H4 with the Npr1 promoter. A, representative levels of acetylated histones H3 and H4 protein expression in cells stimulated with ATRA as analyzed by Western blot. Histone H3 was used as a loading control. ChIP assays demon- strating in vivo recruitment of acetylated histones H3 (B) and H4 (C) to the Npr1 promoter region flanking Ets-1 and Sp1 binding sites. The intensity of DNA bands was quantified by Alpha Innotech analysis software. Representa- tive results of three experiments are shown. WB, Western blot; Ab, antibody.

Article Snippet: Antibodies anti-Ets-1 (N-276), anti-Sp1 (H-225), anti-RAR (C-20), anti-RXR ( N-197), Ac-histone H4 (K-8), histone H3 (FL-136), anti-rabbit IgG-FITC, protein A-agarose, Ets-1 siRNA (mouse), Sp1 siRNA (mouse), RAR siRNA (mouse), RXR siRNA (mouse), and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Western Blot, Control, In Vivo, Binding Assay, Software